The first of two developmental switches in human hemoglobin expression involves the down-regulation or silencing of the embryonic globin gene (e). We have previously reported on a silencer element in the 5' flanking region of the e gene located at -270 bp 5' of the e-globin gene which provides negative regulation for this autonomously regulated gene. We have now extended this analysis 5' up to the globin locus control region (LCR) using reporter gene constructs with deletion mutations and transient transfection assays. Deletion of HS1 had minimal effect on e-globin promoter activity. We identified two negative regulatory regions: eNRA at -3 kb which increases transcription activity by thirty fold in erythroid K562 cells when deleted, and eNRB at -1.7 kb which appears to be erythroid specific. eNRA contains 55% evolutionarily conserved sequences and can reduce transcription activity of a minimal e-globin promoter or the SV40 promoter by two fold or more. Sequence and DNase I footprinting analyses indicate that this region contains a GATA-1 binding motif. Cotransfection of GATA-1 with an e-globin/luciferase construct containing eNRA leads to increased transcription activity, suggesting that the positive effect of GATA-1 dominates over any negative effect associated with the GATA-1 binding motif in the several negative control elements located in this 5' flanking region. For eNRB, DNA binding analyses indicate that a TATA-like binding motif is located within this region. We suggest that the down-regulation of e-globin gene expression as development progresses involves cooperative interaction of the negative regulatory elements, eNRA, eNRB, the e- globin silencer and possibly other negative and positive elements in the 5' flanking region of the e-globin gene.